The Respiratory Syncytial Virus (RSV) is the main causing agent of acute respiratory tract infections in young children worldwide. According to WHO, this virus infects 64 million people yearly, 160.000 of which die (www.who.int). The infection by this virus causes a broad range of clinical symptoms that can be as slight as rhinitis or much more severe such as pneumonia or bronchiolitis, the worst cases being observed in unweaned babies, premature babies, children with congenital cardiopathies and immunodepressed children (1-3).
The infection caused by this virus is utterly frequent and recurrent, since practically 100% of the children younger than 3 years-old have had at least one episode of RSV infection (4). As this infection does not leave a complete immunological memory (5), reinfection is frequent, being less severe as the patient's age increases.
The health situation caused by RSV infection generates a high economical impact for the affected countries. Studies carried out in developed countries estimate that the cost of this infection is over 3.000 euros per patient (6), with a highest limit reaching up to 8.400 euros (6).
The RSV is a negative, unsegmented, single-strand RNA virus with a lipid coat that belongs to the paramyxoviridae virus family, genus pneumovirus (reviewed in (7)). RSV possesses a genome with about 15 kb that encodes for a total of 11 proteins. Five of these proteins have structural functions, corresponding to the transmembrane F, G and SH proteins, the nucleocapsid N protein and the matrix M protein. The other four proteins, M2-1, M2-2, P and L are involved in viral replication and transcription. The remaining two proteins, called NS1 and NS2, are non-structural proteins and they seem to be involved in virulence (8).
The RSV that infects humans has different strains or subgroups, being subgroups A and B those that predominate in the population (reviewed in (9)). The main antigen difference between the subgroups is related to protein G, which only conserves 40-44% of its amino acids between different subgroups (9).
The first vaccine against RSV was tested in the 1960's and comprised a formalin-inactivated complete virus (RSV-FI) that was administered intramuscularly in the presence of alum adjuvant (10). Contrarily to the expected result, this immunization caused a much more severe respiratory case after RSV infection in the vaccinated children, which led to hospitalization of 80% of them and two deaths (11). The respiratory-pulmonary clinical symptoms presented by the vaccinated children were characterized by an unusual eosinophil and neutrophil infiltration together with a high titer of complement-fixing antibodies (11). The analysis of the affected pulmonary tissues of the children vaccinated with RSV-FI that died due to RSV infection showed complement deposition, immune complexes and eosinophil presence in peribronchial regions (12). Together with this, animal studies demonstrate that vaccination with RSV-FI produces a Th2-type immune response, based on T CD4+ lymphocytes, that possesses the same characteristics than those observed in animals that have been immunized with protein G (13) or that receive T CD4+ lymphocytes specific for this protein prior to RSV infection. For this reason, to formulate an effective and safe vaccine against RSV it has been necessary to study thoroughly the immune response generated against the different proteins of this virus, with the aim of identifying those that are able to induce a Th1-type immune response based on interferon-gamma (IFN-γ)-producing and cytotoxic T-lymphocytes.
The current research on vaccines against RSV has been focused on the analysis and development of viral subunits, such as proteins F (14), M2 (15) and also certain conserved protein G segments (16). On the other hand, the production of vaccines based on mutant RSV virus strains, such as those sensitive to temperature (17), having deletions in certain genes (18) or recombinant for cytokines such as GM-CSF (19), has also been studied. Some of these vaccines have been tested in Phase-I and II clinical trials, with variable results (20-22). Other putative vaccine against RSV is represented by the group of vaccines based on proteins F and G, which are administered with adjuvants such as ISCOMs. The immunization with this type of vaccines produces an increased eosinophil infiltration in the pulmonary tissue when a new viral infection develops (23), which increases the damage to the pulmonary tissue.
The immune system of a young child is characterized by developing preferentially Th2-type immune responses, which is possibly caused by the immaturity of the immune system during the first six months of life (24, 25). Nevertheless, if adequately stimulated, the immune system can present a Th1-type response (26). To formulate an effective and safe vaccine against RSV it has been necessary to study thoroughly the immune response generated against the different proteins of this virus, with the aim of identifying those that are able to induce a Th1-type immune response based on cytotoxic T-lymphocytes. The use of bacterial vectors for heterologous expression of viral antigens has the advantage that these bacterial vectors can be used as attenuated living vectors, as they have intact invasion abilities and are recognized as non-pathogenic. An additional advantage of certain bacterial vectors used to express heterologous antigens is their known ability to induce Th1-type immunity (27, 28) which is very attractive for the case of the development of vaccines against RSV (29). The Calmette-Guérin bacillus (BCG) is an attenuated Mycobacterium bovis strain that is used as a vaccine against Mycobacterium tuberculosis in newborns. From the approval of BCG as a vaccine against tuberculosis, this has been administered to more than 3.3 trillion people worldwide. Its massive use has been facilitated by many advantageous characteristics of these bacteria, such as their high thermostability in freeze-dried form. Moreover, the immunization of newborn children with this bacterium is risk-free and only produces minimal side effects. BCG is highly immunogenic and only one dose is able to generate an immune response that is maintained for long terms. Importantly, BCG induces a potent Th1-type immune response both in adults and children (30). This phenomenon is evidenced in newborns by the cell-type immune response generated against antigens of M. tuberculosis (PPD), which is able to survive during extended periods (30).
Up to this date, several bacterial, parasite and viral antigens have been successfully expressed in this bacterial system, which were demonstrated to induce humoral and cellular immunity against these antigens when evaluated in animal models (31, 32). Moreover, BCG has the particularity of not being neutralized by antibodies present in maternal milk, and therefore it can be used as immunity inducer in unweaned babies. The present invention relates to an immunological formulation that comprises one or more attenuated Mycobacterium bacterial strains, preferably the BCG strain, recombinant for RSV proteins, and can be used to prepare vaccines against this virus.
This formulation is directed to avoid or attenuate the pulmonary damage caused by a RSV infection, thanks to the generation of an efficient immune response that is favorable to eliminate the virus. Since attenuated Mycobacterium strains such as BCG are potent Th1-type immune response inducers, the immune response induced by BCG strains recombinant for RSV favors a protection against infections caused by this virus.